PROJECT SUMMARY Despite the widening availability of antiretroviral therapy (ART), almost 2 million people were newly infected with HIV-1 in 2016. While ART is highly effective at controlling viral replication, it does not eliminate infected cells and consequently cannot cure HIV infection. The absence of a cure or effective vaccine is a major impediment to achieving the goal of ending the HIV epidemic. The greatest obstacle to curing HIV is the presence of a reservoir of latently infected cells, which persists for the lifetime of an infected individual despite continuous administration of ART. Precise characterization of the latent reservoir is complicated by a number of factors. Chief among these is distinguishing defective proviruses, which constitute the vast majority of integrated HIV DNA and are incapable of reactivation, from replication-competent proviruses, which are the true target of HIV eradication strategies. The most commonly used method to investigate the latent reservoir, the sequencing of short segments of proviral HIV DNA, cannot distinguish between replication-competent and replication-incompetent viruses. Previous approaches that have been utilized to isolate replication-competent latent viruses typically identified a single virus, or viruses with very limited diversity. To more sensitively and precisely analyze the viral sequences in the replication-competent reservoir, we developed and published a novel technique, termed the qualitative and quantitative viral outgrowth assay. Using this assay, we demonstrated for the first time that the replication-competent reservoir is genetically and phenotypically diverse, and is comprised of groups of clonal sequences. We now propose to investigate the diversity of the replication-competent reservoir in clinical trials to determine the origins of viral rebound and the stability of the latent reservoir. The central hypothesis of this proposal is that the diversity of the replication-competent reservoir can be explored to address fundamental questions about the composition, location, and dynamics of the latent reservoir. Our specific aims are: to determine if rebound viremia originates from the lymphoid tissue or peripheral blood reservoirs and to compare the diversity of the reservoirs within these compartments; to determine if viral rebound during treatment with the broadly neutralizing antibody 3BNC117 changes the genetic composition of the reservoir; and to evaluate the ability of the combination of the latency-reversing agent romidepsin and the broadly neutralizing antibody 3BNC117 to alter the genetic composition of the latent reservoir. The expected outcomes of the above aims will expand our understanding of the composition of the reservoir and the origin of viral rebound, and identify new research directions to pursue in an R01 proposal to further characterize the latent reservoir and assess novel strategies to eradicate it.